JPH11180894A - Agent for antithrombotic treatment or prophylaxis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an agent useful for the antithrombotic treatment or prophylaxis. SOLUTION: This agent comprising a chimeric immunoglobulin or an immunological fragment containing a nonhuman-derived antigen binding region specific for glycoproteins IIb/IIIa and a human constant region and having the specificity for the glycoproteihs IIb/IIIa is useful for the treatment or prophylaxis of thrombosis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] background platelet aggregation of the invention is an essential event in the formation of the blood clot. Under normal circumstances, blood clots act to prevent blood cells from escaping from the vasculature. However, during certain disease states (eg, myocardial infarction), clots restrict blood flow or block it altogether, resulting in necrosis of cells.

[0002] Patients with a heart attack are typically treated with a thrombolytic agent such as tissue plasminogen activator or streptokinase, which dissolves the fibrin component of the clot. The major complication associated with fibrinolysis is reocclusion due to platelet aggregation, which further results in cardiac damage. Since the glycoprotein (gp) IIb / IIIa receptors are known to be responsible for platelet aggregation, reagents that block these receptors reduce or prevent reocclusion following thrombolysis and It is expected to accelerate the rate of dissolution.

One method for inhibiting platelet aggregation involves a monoclonal antibody specific for the gpIIb / IIIa receptor. A murine monoclonal antibody designated 7E3 that inhibits platelet aggregation and appears to be useful in the treatment of human thrombosis has been published in published European Patent Applications 205,270 and 206,532.
No. It is known in the art that murine antibodies have severe limitations when used in human therapy. As a foreign protein, murine antibodies may reduce or destroy their therapeutic effect and / or provoke an immune response that elicits an allergic or hypersensitivity reaction in the patient. The need for re-administration of such therapeutic modalities in thromboembolism increases the likelihood of these types of immune responses.

[0004] Chimeric antibodies consisting of a non-human binding region linked to a human constant region have been suggested as a means of preventing the problem of the murine antibody's immune response. PNAS , 81: 685
1 (1984) and PCT application PCT / GB85003
See 92. Since the constant region is largely responsible for the immunoreactivity of the antibody molecule, chimeric antibodies having constant regions of human origin will be less likely to elicit an anti-murine response in humans. However, it is unpredictable whether linking a human constant region to a murine binding region of the desired specificity would reduce immunoreactivity and / or alter the binding capacity of the resulting chimeric antibody.

SUMMARY OF THE INVENTION The present invention is directed to a platelet-specific chimeric immunoglobulin comprising a variable region or antigen-binding region of specific non-human origin and a constant region of human origin. The chimeric immunoglobulin is g
It can be specific for the pIIb / IIIa receptor or other platelet components. These antibodies can bind to platelets and inhibit platelet aggregation, and are thus useful as antithrombotic agents and to prevent or reduce reocclusion following thrombolysis.

DETAILED DESCRIPTION OF THE INVENTION The chimeric immunoglobulins of the present invention comprise individual chimeric heavy and light immunoglobulin chains. The chimeric H chain has gpIIb linked to the human H chain constant region.
/ IIIa comprising an antigen-binding region derived from the heavy chain of a non-human antibody specific for the receptor. The chimeric L chain is human L
The antigen-binding region derived from the light chain of the non-human antibody linked to the chain constant region.

[0007] The immunoglobulins of the present invention can be monovalent, divalent or polyvalent. Monovalent immunoglobulin is a dimer (HL) formed from a chimeric heavy chain linked to a chimeric light chain via a disulfide bridge. Bivalent immunoglobulins are tetramers (H ₂ L ₂ ) formed from two dimers linked via at least one disulfide bridge. Polyvalent immunoglobulins can be produced, for example, by using aggregated heavy chain constant regions (eg, IgMH chains). Fab, Fab 'or F (ab') ₂
Chimeric immunoglobulin fragments can also be produced.

[0008] The non-human antigen binding region of the chimeric immunoglobulin is derived from an immunoglobulin specific for platelets. A preferred immunoglobulin is platelet gpII
Blocks ligands that are specific for the b / IIIa receptor and bind to the glycoprotein gpIIb / IIIa receptor complex. Examples of suitable antibodies include 7E3 and 10E5. European Patent Application No. 205,2
No. 70 and No. 206,532. These teachings are incorporated herein. The 7E3 antibody (or an antibody reactive therewith or a functionally equivalent epitope) is gpIIb / I
Specific for the complexed form of the IIa receptor,
Particularly preferred. Other antibodies specific for either the IIb or IIIa component can also be used. Antibodies specific for other platelet antigens can be used. For example, the S12 antibody [Journal of Biological Chemistry (J.
Biol. Chem), 259 : 9799-9804.
(1984)] GMP-14
0 can be used.

[0009] Preferably, the antigen binding region is of murine origin. The reason is that platelets, especially gpIIb /
This is because murine antibodies against the IIIa receptor are available or can be produced in murine systems. Other animals or rodent species provide another source of the antigen binding region.

[0010] The constant region of the chimeric antibody is derived from human immunoglobulin. The heavy chain constant region can be selected from any of the five isotypes α, β, ε, γ, or mu. Furthermore, various subclasses of H chains (H chain Ig
(Like the G subclass) are responsible for different effector functions and thus by choosing the desired heavy chain constant region,
A chimeric antibody having a desired effector function can be produced. A preferred constant region is γ1 (IgG
1), γ3 (IgG3) and γ4 (IgG4).
The light chain constant region can be of the kappa or lambda type.

In general, a chimeric antibody is a platelet-specific, non-human source linked to a second DNA segment encoding at least a portion of the human constant region for each of the light and heavy chain components of the chimeric immunoglobulin. Produced by producing a fusion gene comprising a first DNA segment encoding at least a functional portion of a target variable region (eg, a functionally rearranged variable region with a joining segment). Each fused gene is integrated or inserted into an expression vector. Recipient cells are
Recipient cells capable of expressing the gene product are then transfected with the gene. The transfected recipient cells are cultured under conditions that allow expression of the introduced gene, and the expressed immunoglobulin or immunoglobulin chain is recovered.

[0012] The genes encoding the variable regions of the Ig L and H chains can be obtained from lymphocytes that produce platelet-specific antibodies. For example, gpIIb / IIIa
Hybridoma cell lines that produce antibodies to the receptor provide the source of the immunoglobulin variable regions of the chimeric antibodies of the invention. Other rodent cell lines are available. The cell line may be a rodent prepared by converting human platelets or gpIIb /
IIIa receptor-containing components or fractions of platelets, challenged to form hybrid cells fused with the antibody-producing cells and the myeloma cell line, cloned the hybrids, and purified platelets or the glycoprotein gpIIb / II
It can be produced by selecting a clone producing an antibody against the Ia receptor.

[0013] The constant region can be obtained from human antibody-producing cells by standard cloning techniques. Alternatively, constant regions of human origin are readily available from these clones, as genes representing two classes of light chains and five classes of heavy chains have been cloned. F (a
b ′) Chimeric antibody binding fragments such as ₂ and Fab fragments can be produced by designing the chimeric H chain gene in a truncated form. For example, a chimeric gene encoding a F (ab ') ₂ H chain portion would include DNA sequences encoding the CH ₁ domain and hinge region of the H chain.

[0014] Preferably, the fused genes encoding the L and H chimeric chains (or portions thereof) are incorporated into two different expression vectors which can be used to co-transfect recipient cells. . Each vector contains two selectable genes--one for selection in bacterial systems and one for selection in eukaryotic cell systems--each vector has a different gene pair. . These vectors allow the production and amplification of the fusion gene in a bacterial system and the subsequent co-transfection of eukaryotic cells and the selection of co-transfected cells. Examples of selectable genes for bacterial systems are those that confer ampicillin resistance and those that confer chloramphenicol resistance. Two selectable genes for the selection of eukaryotic transfectants are (i) the xanthine-guanine phosphoribosyltransferase gene (g pt ) and (ii) the phosphotransferase gene from Tn5 ( ne
o ) is preferred. Selection by gpt is based on the ability of the enzyme encoded by this gene to use xanthine as a substrate for purine nucleotide synthesis, and a similar endogenous enzyme cannot be selected. Only cells expressing the gpt gene can survive in a medium containing xanthine and mycophenolic acid, which blocks the conversion of inosine monophosphate to xanthine monophosphate. The product of neo is the antibiotic G41
Blocks the suppression of protein synthesis in eukaryotic cells caused by 8 and other antibiotics in its class. The two selection methods can be used simultaneously or sequentially to select for expression of an immunoglobulin chain gene introduced on two different DNA vectors into a eukaryotic cell.

A preferred recipient cell line is a myeloma cell. Myeloma cells are transfected with I
The immunoglobulin encoded by the g gene can be synthesized, assembled and secreted. In addition, they have a mechanism of glycosylation of immunoglobulins. Particularly preferred recipient cells are Ig non-producing myeloma cells Sp2
/ 0. The cells produce only immunoglobulins encoded by the transfected immunoglobulin genes. Myeloma cells can be grown in culture or in the peritoneum of mice, where secreted immunoglobulins can be obtained from ascites. Other lymphocytes, such as B lymphocytes or hybridoma cells, can serve as suitable recipient cells.

There are several ways to transfect lymphocytes with a vector containing an immunoglobulin-encoding gene. A preferred method of introducing DNA into lymphocytes is by electroporation. In this method, recipient cells are exposed to an electric pulse in the presence of the DNA to be introduced. For example, Potter et al., PNAS 81 :
7161 (1984). Another method of introducing DNA is by protoplast fusion. In this method, lysozyme is used to peel cell walls from bacteria containing a recombinant plasmid containing a chimeric Ig gene. The resulting protoplasts are fused with myeloma cells by polyethylene glycol. After protoplast fusion, transfectants are selected and isolated. Another method that can be used to introduce DNA into many types of cells is calcium phosphate precipitation.

The chimeric immunoglobulin gene can be expressed in non-lymphocytes such as bacteria or yeast. When expressed in bacteria, immunoglobulin heavy and light chains become part of inclusion bodies. Thus, the chain
It must be isolated, purified, and then assembled into a functionalized immunoglobulin molecule.

The chimeric platelet-specific antibody of the present invention is useful as a therapeutic agent for antithrombosis. Chimeric antibodies (or fragments thereof) can be used to inhibit platelet aggregation and thrombus formation. This antibody can be used in any situation where thrombus formation or remodeling is to be prevented. For example, the antibody can be used alone to prevent clots in post-angioplasty procedures, pulmonary embolism, deep vein thrombosis and coronary bypass surgery. The antibody can be administered with a thrombolytic agent, such as tissue plasminogen activator, urokinase or streptokinase, to prevent or reduce possible reocclusion after thrombolysis and promote clot lysis. The antibody or fragment can be administered before, together with, or after administration of the thrombolytic agent in an amount sufficient to prevent platelet aggregation that can cause re-occlusion. The antibodies are given parenterally, preferably intravenously, in a pharmaceutically acceptable vehicle, such as sterile saline. Antibodies can be given in multiple doses or by controlled release mechanisms (eg, via polymer or batch administration systems).

[0019] During repeated treatment with anti-platelet antibodies, drug-induced thrombocythemia can occur, which causes the body to recognize antibody-coated platelets as foreign proteins that give rise to antibodies against them and then recognize them. The result may be faster removal than usual. The use of chimeric anti-platelet (eg gpIIb / IIIa) antibodies can circumvent this problem. Most of the murine components of the chimeric antibody are platelets (eg, gpIIb / IIIa receptor)
And thus would be rejected by the immune system making the chimeric antibody functionally distinguishable from the same epitope-directed human antibody and would therefore be expected to be non-immunogenic.

The platelet-specific chimeric immunogenic globulin of the present invention is also useful for thrombus imaging. For this purpose, antibody fragments are generally preferred. As described above, the chimeric heavy chain gene is designed in a truncated form to produce a chimeric immunogenic globulin fragment (eg, Fab, Fab ′ or F (a) for imaging of immunogenic scintigraphy.
b ′) ₂ ) can be generated. These molecules are
Labeling with a radioisotope, such as ¹³¹ Iodine, ¹²⁵ Iodine, ^99m Technetium or ¹¹¹ Indium, either directly or through a coupled chelating agent such as DTPA, can produce a radioimmunogenic scintigraphic agent. . Alternatively, a radiometal binding (chelating) domain can be engineered into the chelating antibody site to provide a labeling site. Thus, chelated immunogenic globulin can be designed as a protein having a non-human platelet-specific variable region, a human constant region (preferably truncated) and a metal binding domain from a metal binding protein such as metallothionein.

The platelet-specific chelating immunogenic globulin is administered to a patient suspected of having a thrombus. After a time sufficient to localize the labeled immunogenic globulin to the site of the thrombus, the signal generated by the label is detected by a photoscan device such as a gamma camera. The detected signal is then converted to a thrombus image. This image makes it possible to locate the thrombus in vivo and to devise an appropriate treatment method.

The present invention is further described by the following examples. Unless otherwise specified, all parts and percentages are by weight and degrees are Celsius.

Description Example 1 Production of Chimeric Platelet-Specific IgG4 A. General Method A method for cloning the variable regions for the heavy and light chain genes from the 7E3 hybridoma was described as being functionally sequenced (and expressed). A) Genomic ligation based on the variable region for the Ig gene and the corresponding J (ligation) region. A genomic library is screened using the J region DNA probe to detect the DN linked to the J region.
A can be isolated. Reproductive system configuration (germli
DNA in ne configuration (not rearranged) also hybridizes to the J probe, but is not ligated to the variable region sequence, and can be identified by restriction analysis of the isolated clone.

[0024] Thus, the cloning method, J _H and J _K
The purpose was to isolate variable regions from rearranged heavy and light chain genes using probes. These clones were tested by Northern analysis to determine if their sequences were expressed in the 7E3 hybridoma. These clones containing the expressed sequences were placed in an expression vector containing the human constant region and transfected into mouse myeloma cells to determine if the antibody was produced. Antibodies from productive cells were then tested for binding specificity and functionality as compared to the 7E3 murine antibody.

B. Materials and Methods Construction of H Chain Genome Library To isolate the H chain variable region gene from the 7E3 hybridoma, a size selection genomic library was constructed using the phage λ vector gt10. Southern analysis of the EcoRI-digested 7E3 DNA using the _JH probe showed a single 3.5 kb band corresponding to the rearranged heavy chain positions. This fragment appeared to contain the 7E3 heavy chain variable region gene. High molecular weight DNA was isolated from 7E3 hybridoma cells and completely digested with the restriction endonuclease EcoRI. The DNA was then fractionated on a 0.7% agarose gel and DNA in the 3-4 kb size range was isolated directly from the gel. After phenol / chloroform extraction and Sephadex G-50 gel filtration, the 3-4 kb fragment was ligated with the λGT10 arm (Promega Biotech, Inc.) and phage ex vivo using packageene from Promega Biotech. Packaged in particles. The library was screened directly at a density of approximately 30,000 plaques / 150 mm petri dish using a ³² P-labeled J _H probe. Plaque hybridization is 5 × SS
C, 50% formamide, 2 × Denhardt's reagent, 20
18-2 at 42 ° C. in 0 μg / ml denatured salmon sperm DNA
Performed for 0 hours. Final wash: 0.5 x SSC, 0.1% S
Performed at 65 ° C. in DS. Positive clones were identified after autoradiography.

L chain genomic library construction To isolate the variable region gene of the 7E3 L chain, a genomic library was constructed in the λ vector EMBL-3.
High molecular weight DNA was partially digested with the restriction endonuclease Sau3A and size fractionated on a 10-40% sucrose density gradient. The DNA fragment of 18-23 kb was EM
It was ligated with the BL-3 arm and packaged in vitro into phage particles using a packager. This library was screened at a density of 30,000 plaques / 150 mm plate using a J _K probe.
Hybridization and washing conditions were the same as used for the heavy chain library.

DNA Probe The mouse H chain J _H probe is a 2 kb BamHI / EcoRI fragment containing both J3 and J4 segments. Mouse L chain J _K probe is a HindIII fragment of 2.7kb including all five J _K segments.
^{The 32} P-labeled probe was manufactured by nick translation using a kit obtained from Amersham. Free nucleotides were removed by centrifugation through a Sephadex G-50 column. The specific activity of the probe was about 10 ⁹ cpm / μg.

Northern analysis 15 μg of total cellular DNA was subjected to electrophoresis on a 1% agarose / formaldehyde gel (Maniatis et al., Molecular Cloning) and transferred to nitrocellulose. Blots were hybridized with 50% formamide, 2 × Denhardt's reagent, 5 × SSC and nick-translated DNA probe in 200 μg / ml denatured salmon sperm DNA at 42 ° C. for 10 hours. The final washing condition is 0.5 × SSC 0.1% S at 65 ° C.
DS.

DNA using electroporation
Transfection Plasmid DNA to be transfected is
2 until equilibrium in ethidium bromide / cesium chloride gradient
Purified by centrifugation twice. 10-50 μg of plasmid DNA was added to 8 × 10 ⁶ SP2 in PBS on ice.
/ 0 cells and the mixture was placed in a Bio-Rad electroporation apparatus. Electroporation was at 200 volts and cells were attached to a 96-well microtiter plate (plate ou).
t). Appropriate drug selection was applied after 48 hours and drug resistant colonies were identified after 1-2 weeks.

Using a standard curve obtained with quantitatively purified IgG for antibody production , a particle concentration fluorescence immunoassay (Jolly M.E. et al., Jar.
Null of Immunological Methods , 67:21 )
The concentration of the chimeric 7E3 antibody with human constant regions analyzed for the IgG protein content of the tissue culture supernatant was determined using goat anti-human IgG Fc antibody coated polystyrene beads and fluorescein-conjugated goat anti-human IgG Fc antibody. The assay was performed on an automated instrument (Pandex Laboratories).

Purification of Platelet-Specific Chimeric IgG4 Antibody Tissue culture supernatants were concentrated on a Diaflo YM100 ultrafiltration membrane (Amicon) and applied to a protein A-Sepharose column. The chimeric antibody was eluted from the protein A column by a sodium citrate pH gradient from pH 6.5 to pH 3.5. Purify the antibody with Diaflo Y
Concentrate using a M100 ultrafiltration membrane. Antibody concentration is 2
It was determined by determining the absorbance at 80 nm.

Binding Inhibition Assay The purified antibody (murine 7E3 or chimeric 7E3) was used to detect radioiodinated 7 for binding to human platelets.
Competed with E3 antibody. Platelet-rich plasma (PRP)
Was prepared by centrifuging citrated whole human blood for 3.5 minutes at 1875 rpm. ¹²⁵ I label 7E
Three antibodies (150,000 cpm) were added to the appropriate dilutions of the purified test antibody and the reaction was started by the addition of 150 μl PRP. Incubate at room temperature 1-
Platelets with the antibody bound for 2 hours and free antibody were removed from the free antibody at 12,000 in a 0.4 ml microfuge tube.
Separated by centrifugation through 30% sucrose at 0 g for 4 minutes. The tube tip containing the platelet / antibody pellet was cut off and counted in a gamma counter. Iodized 7E
The competition of platelets 3 and chimera 7E3 for binding to platelets was compared to that of iodinated 7E3 and unlabeled 7E3 IgG.

Inhibition of Platelet Aggregation Purified 7E3 or chimeric 7E3 antibody was added to citrated whole human blood and incubated at 37 ° C. for 10 minutes. The rate of platelet aggregation was measured after activation with collagen or ADP using a whole blood agglutination detector (Chronolog).

C. Results Cloning of Platelet-Specific Variable Gene Region Each of the positive clones from the heavy and light chain libraries was screened after screening about one million plaques using the J _H and J _K probes, respectively. Isolated. After at least three rounds of plaque purification, bacteriophage DNA was isolated for each positive clone and EcoRI
(H chain clone) or HindIII (L chain clone)
And fractionated on a 1% agarose gel. DNA
To nitrocellulose and blot to J _H (H
Chain) or J _K ³² were P labeled DNA probe hybridized. 2 containing the 3.5kbEcoRIDNA fragment hybridized to J _H probe
Two clones were obtained. 3.0 and two sizes HindIII fragment class 6.0kb was identified by J _K probe.

The cloned DNA corresponding to the intrinsic heavy and light chain variable regions from the 7E3 hybridoma was:
It should hybridize to mRNA isolated from the hybridoma. Non-functional DNA rearrangements at the heavy or light chain positions should not be expressed. FIG.
3.5 kb EcoRI putative heavy chain fragment and 6.0 kb Hin
Shown is Northern analysis showing that each dIII putative light chain fragment hybridizes to an appropriately sized mRNA from the 7E3 hybridoma. The subcloned fragment was labeled with ³² P by nick translation and hybridized to a Northern blot containing total RNA from SP2 / 0 (7E3 hybridoma fusion partner) or 7E3. 3.5 kb EcoR
The IH chain fragment hybridizes to 2 kb mRNA of 7E3 RNA, but not to SP2 / 0 RNA. Similarly, the 6.0 kb L chain HindIII fragment is:
Hybridizes with 1250 bp mRNA in 7E3 RNA, but not in SP2 / 0 RNA. These are the exact sizes of the heavy and light chain mRNAs, respectively. The cloned DNA fragment was 7E3
These data suggest that these are the correct variable region sequences from the 7E3 hybridoma, as they include sequences expressed in the hybridoma. However, final functional studies demonstrate that these sequences, when combined with the appropriate constant region sequences, can direct the synthesis of antibodies having similar specificity and affinity as the murine 7E3 antibody. It is.

Vector and Expression System The putative L and H chain V genes cloned from the 7E3 hybridoma were described above (San L. et al., PNAS).
84, pp. 214-218 (1987)) and linked to human K and G4 constant region genes in an expression vector.
pSV184 of △ Hneo17-1AV _K hC _K 17-1
The AV _K HindIII fragment was replaced with a 6.0 kb HindIII fragment corresponding to the putative light chain variable region gene from 7E3. Similarly, pSV2 @ Hgpt17-1A
The 17-1AV _H EcoRI fragment of V _H -hC _G4, 7E3
3.5 kb Ec corresponding to the putative H chain V region gene from
Replaced with the oRI fragment. p7E3V _K hC _HK and p7
The structure of the plasmid obtained was named E3V _H hC _G4 shown in FIG.

To express the chimeric heavy and light chain genes, the two plasmids were co-transfected into the non-producing mouse myeloma cell line SP2 / 0. Light chain plasmids confer resistance to G418 and heavy chain plasmids confer resistance to mycophenolic acid, thus allowing the use of double selection to obtain clones carrying and expressing genes from each plasmid. And Colonies resistant to G418 and mycophenolic acid were expanded to stable cell lines and maintained in the presence of both drugs. Tissue culture supernatants from these cell lines were tested for antibody using a particle concentration fluorescence immunoassay with polystyrene beads coated with a goat anti-human IgG Fc antibody and the same antibody labeled with fluorescein. From the first 10 lines checked, about 2 μg / ml
The one that produced (named c-7E3F6) was selected for further consideration.

Platelet Binding Activity Assay c-7E Using Protein A-Sepharose Column
After purification of the 3F6 antibody, the antibody was concentrated and compared to murine 7E3 IgG in a platelet binding activity assay. FIG. 3 shows that murine 7E3 and c-7E3F6 (a putative chimeric antibody) radiolabeled 7 to the same extent for platelet binding.
Shows that it competes with E3. The binding curves can overlap, indicating that the binding characteristics of murine and chimeric 7E3 are the same.

Inhibition of Platelet Aggregation by c-7E3F6 Purified c-7E3F6 was compared to murine 7E3 in a functional assay that measures the ability of the test antibody to inhibit human platelet aggregation. The results shown in FIG. 4 show that both antibodies inhibit collagen-induced platelet aggregation to the same extent at the same antibody concentration. c-7E3F6 also suppresses ADP-induced platelet aggregation to a similar extent.

The results of the platelet binding assay and platelet aggregation inhibition assay were as follows: ) That the correct variable region gene is actually cloned from the 7E3 hybridoma;
2) Replacing the mouse constant region with the human constant region indicates that it has no effect on the binding or functional properties of the 7E3 variable region as measured by these assays.

Fibrogen-Coated Bead Assay Chimeric c-7E3F6 antibody was found to be positive in a qualitative, functional assay that measures the ability of the antibody to inhibit aggregation between platelets and fibrinogen-coated beads. (1983) Journal of Clinical Investigation (J. Cli)
n. Invest), 73 : 325-338.

Example 2 Chimeric IgG1 and IgG3
The DNA segment encoding the variable region of the heavy chain from the manufactured murine 7E3 antibody was
By replacing with the E3 variable region fragment, the expression vectors pSV2ΔHgpt17-1AV _H -hC _G1 and pS
V2 @ Hgpt17-1AV _H -hC _G3 (Sun et al., PN
AS 84, pp. 214-218, 1987). Chimera H obtained
The chain gene was co-transfected with the chimeric light chain gene into SP2 / 0 cells to produce γ1, K and γ3,
A stable cell line secreting the K antibody was generated.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, equivalents to the embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[Brief description of the drawings]

FIG. 1 shows the results of Northern analysis of H and L chain mRNA's for a 7E3 monoclonal antibody using the cloned variable region as a probe.

FIG. 2. Plasmid p7 with chimeric 7E3 immunoglobulin light and heavy chain chimeric gene constructs, respectively.
It is a schematic representation of the E3V _K hC _k and p7E3V _H hC _G4.

FIG. 3 is a graph showing the binding of chimeric 7E3 immunoglobulin to platelets.

FIG. 4 is a graph showing suppression of platelet aggregation by chimeric 7E3 immunoglobulin.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12N 15/02 A61K 37/54 // C12P 21/08 C12N 15/00 C (72) Inventor Barry S. Koller New York State 11746 Dixhills Durham Drive 2 (72) Inventor David M. Knight 19301 Paoli Fezantlan Drive, Pennsylvania, USA 208

Claims (21)

[Claims] 1. An immunoglobulin or fragment comprising, as an active ingredient, a chimeric immunoglobulin or immunoglobulin fragment comprising an antigen-binding region of non-human origin specific for glycoprotein IIb / IIIa and a human constant region. Has specificity for glycoprotein IIb / IIIa. 2. The agent according to claim 1, wherein the antigen-binding region of non-human origin is of murine origin. 3. The glycoprotein IIb / III wherein the chimeric immunoglobulin or chimeric immunoglobulin fragment is a ligand.
2. The agent according to claim 1, which is capable of inhibiting binding to a receptor. 4. The monoclonal antibody 7E whose antigen-binding region is
3. The drug according to claim 1, which is derived from No. 3. 5. An immunoglobulin fragment comprising Fab, Fab ′
Or the F (ab ') ₂ fragment. 6. The agent of claim 1, wherein the chimeric immunoglobulin or chimeric immunoglobulin fragment is capable of competing with a 7E3 monoclonal antibody for binding to human platelets. 7. A chimeric immunoglobulin or immunoglobulin fragment comprising (i) a thrombolytic agent, and (ii) an antigen-binding region of non-human origin specific for glycoprotein IIb / IIIa and a human constant region. Contained as an active ingredient, wherein the immunoglobulin or fragment is glycoprotein IIb /
A pharmaceutical composition for treating or preventing antithrombosis, which has specificity for IIIa. 8. The pharmaceutical composition for the treatment or prevention of antithrombosis according to claim 7, wherein the antigen-binding region of non-human origin is of murine origin. 9. The pharmaceutical composition for antithrombotic treatment or prevention according to claim 7, wherein the thrombolytic agent is tissue plasminogen activator, streptokinase or uroxase. 10. The glycoprotein IIb / II wherein the chimeric immunoglobulin or chimeric immunoglobulin fragment is a ligand.
The pharmaceutical composition for antithrombotic treatment or prevention according to claim 7, which is capable of inhibiting binding to an Ia receptor. 11. The method according to claim 11, wherein the antigen-binding region is a monoclonal antibody 7.
The pharmaceutical composition according to claim 7, which is derived from E3. 12. An immunoglobulin fragment comprising Fab, Fa
The pharmaceutical composition for antithrombotic treatment or prevention according to claim 11, which is b 'or F (ab') ₂ fragment. 13. The pharmaceutical composition according to claim 7, wherein the chimeric immunoglobulin or chimeric immunoglobulin fragment is capable of competing with a 7E3 monoclonal antibody for binding to human platelets. 14. A chimeric immunoglobulin or immunoglobulin fragment comprising a non-human antigen-binding region specific to glycoprotein IIb / IIIa and a human constant region as an active ingredient, said immunoglobulin or fragment thereof. Has specificity for glycoprotein IIb / IIIa for administration to a patient having a thrombus or at risk of thrombus formation together with a thrombolytic agent. 15. The agent according to claim 14, wherein the antigen-binding region of non-human origin is of murine origin. 16. The medicament according to claim 14, wherein the administration of the medicament is before, simultaneously with, or after administration of the thrombolytic agent. 17. The agent according to claim 14, wherein the thrombolytic agent is tissue plasminogen activator, streptokinase or uroxase. 18. The glycoprotein IIb / II wherein the chimeric immunoglobulin or chimeric immunoglobulin fragment is a ligand
15. The method according to claim 14, wherein the binding to the Ia receptor can be prevented.
The drug as described. 19. The method according to claim 19, wherein the antigen-binding region is a monoclonal antibody 7.
The drug according to claim 14, which is derived from E3. 20. An immunoglobulin fragment comprising Fab, Fa
The agent according to claim 19, which is a b 'or F (ab') ₂ fragment. 21. The method of claim 14, wherein the chimeric immunoglobulin or chimeric immunoglobulin fragment is capable of competing with a 7E3 monoclonal antibody for binding to human platelets.